The present invention relates to novel methods and products directed to immunosuppression via the inhibition of cathepsin S. The methods and products may by employed for the treatment of autoimmune diseases, as well as reducing the competency of class II MHC molecules for binding antigenic peptides.
Class II MHC (major histocompatibility complex) cellular proteins (xcex1xcex2 heterodimers) associate early during biosynthesis with a type II membrane polypeptide, the invariant chain (Ii), to form class II MHC/invariant chain complexes (xcex1xcex2Ii). It has been reported that the invariant chain associates with class II MHC molecules via direct interaction of residues 81-104 of its lumenal domain, designated class II associated invariant chain peptides (CLIP), with the antigen binding groove of class II MHC.
The invariant chain contains a signal in its cytoplasmic tail which delivers the class II MHC/invariant chain complexes to intracellular endocytic compartments, where the class II MHC molecules encounter and bind antigenic peptides. A prerequisite for antigenic peptide loading of class II MHC molecules is the proteolytic destruction of the invariant chain from the class II MHC/invariant chain complexes. Identification of the specific key protease responsible for this proteolysis has not previously been reported. Proteolysis of the invariant chain allows the antigenic peptides to bind to the class II MHC molecules to form class II MHC/antigenic peptide complexes.
The antigenic peptides in these complexes are then deposited on the cell surface for recognition by CD4+T cells. These T cells are involved in the production of cytokines and thus help orchestrate an immune response, culminating in the appropriate production of antibodies. On occasion, CD4+cells are activated inappropriately and are believed to contribute to the pathology of autoimmune disease.
In one aspect, the present invention provides methods for inhibiting invariant chain proteolysis from class II MHC/invariant chain complexes, reducing the competency of class II MHC molecules for binding antigenic peptides, and reducing the presentation of antigenic peptide by class II MHC molecules, by administering to a mammalian cell, in vivo or in vitro, an amount of an inhibitor of cathepsin S effective to substantially inhibit proteolysis of invariant chain by cathepsin S.
In another aspect, the present invention provides methods for modulating class II MHC-restricted immune responses. Such immune responses are essential to autoimmune diseases, allergic reactions, and allogeneic tissue rejections. Therefore, the present invention also provides methods for suppressing class II MHC-restricted immune responses and, in particular, autoimmune, allergic, and allogeneic immune responses, by administering to a mammal (e.g., a human patient) a therapeutically effective amount of an inhibitor of cathepsin S to reduce the presentation of antigenic peptides by class II MHC molecules and, thereby, provide a degree of relief from these conditions. In preferred embodiments, methods are provided for the treatment of autoimmune diseases including juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves"" disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto""s thyroiditis. In other preferred embodiments, methods are provided for treating allergic responses, including asthma, and for treating allogeneic immune response, including those which result from organ transplants, including kidney, lung, liver, and heart transplants, or from skin or other tissue grafts.
The inhibitors of cathepsin S may be any molecular species which inhibit the transcription of a cathepsin S gene, the processing or translation of a cathepsin S mRNA, or the processing, trafficking or activity of a cathepsin S protein, when administered in vivo or in vitro to a mammalian cell which is otherwise competent to express active cathepsin S. In particular, inhibitors may be repressors, or antisense sequences, or competitive and non-competitive inhibitors such as small molecules which structurally mimic the natural substrates of cathepsin S but which are resistant to the proteolytic activity of the enzyme, or antibodies, ribozymes, and the like. Preferably, the inhibitors are cysteine protease inhibitors.
In preferred embodiments, the cathepsin S inhibitors are xe2x80x9cselectivexe2x80x9d inhibitors of cathepsin S which fail to inhibit, or inhibit to a substantially lower degree, at least one of cathepsins K, L, H, O2 and B, and in most preferred embodiments, the inhibitors are xe2x80x9cspecificxe2x80x9d inhibitors of cathepsin S which fail to inhibit, or inhibit to a substantially lower degree, each of cathepsins K, L, H, O2 and B.
In addition, preferred inhibitors include peptide-based inhibitors which mimic a portion of a naturally occurring cathepsin S substrate. Such peptide based inhibitors include peptidyl aldehydes, nitriles, xcex1-ketocarbonyls, halomethyl ketones, diazomethyl ketones, (acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts, epoxides, and N-peptidyl-O-acyl-hydroxylamines. Preferred peptide-based inhibitors of cathepsin S also include those based upon the sequences Leu-Leu-Leu, and Leu-Hph, such as Leu-Leu-Leu-vinyl sulfone, N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone, N-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone, and morpholinurea-Leu-Hph-vinylsulfone phenyl (LHVS).
In another aspect, the present invention provides a new class of peptide-based inhibitors of cathepsin S based upon the newly disclosed preferred chain cleavage site spanning from N-terminally about positions 68-75 to C-terminally about positions 83-90 of the invariant chain sequence. Thus, peptide-based inhibitors of cathepsin S based upon a sequence of 2-20, more preferably 2-10, and most preferably 2-3 consecutive residues from within this site are provided. Particularly preferred are peptide-based inhibitors of cathepsin S based upon the sequences Asn-Leu, Glu-Asn-Leu, Arg-Met, and Leu-Arg-Met (positions 77, 78, and 79, or xe2x88x923, xe2x88x922 and xe2x88x921 relative to the Lys80 cleavage point) are preferably used as a basis for choosing or designing a peptide-based inhibitor.
Thus, for example, the invention provides novel peptide-based inhibitors such as vinylsulfone compounds including Asn-Leu-vinylsulfone, Arg-Met-vinylsulfone, Leu-Arg-Met-vinylsulfone, and Glu-Asn-Leu-vinylsulfone. Modifications of these peptide vinylsulfones are also included in the invention. For example, carboxybenzyl can be present at the N-terminal end to give the following compounds: N-(carboxybenzyl)-Asn-Leu-vinylsulfone, N-(carboxybenzyl)-Arg-Met-vinylsulfone, N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone, and N-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone. In an alternative, nitrophenylacetyl is present at the N-terminal end to give the following compounds: N-(nitrophenylacetyl)-Asn-Leu-vinylsulfone, N-(nitrophenylacetyl)-Arg-Met-vinylsulfone, N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone, and N-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone.
The invention is also meant to include other peptide-based inhibitors based on the peptide sequences of the preferred invariant chain cleavage site of cathepsin S, including peptidyl aldehydes, nitriles, xcex1-ketocarbonyls, halomethyl ketones, diazomethyl ketones, (acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts, epoxides, and N-peptidyl-O-acyl-hydroxylamines, and those with various other substitutions at the amino terminus as would be known to those skilled in the art.
Other embodiments of the present invention will be apparent to one of ordinary skill is in the art from the foregoing and from the Detailed Description and Examples presented below.
The present invention is based, in part, upon the discovery that the mammalian cysteine protease cathepsin S is of primary importance in the proteolysis of invariant chain polypeptides complexed to class II ARC xcex1xcex2 heterodimers. In particular, it is herein disclosed that cathepsin S appears to be responsible for the normal cleavage of the invariant chain polypeptide while it is associated in class II MHC/invariant chain complexes and, therefore, that inhibition of cathepsin S inhibits the proteolysis of invariant chain from class II MHC/invariant chain complexes are inhibits the formation of class II MHC/CLIP complexes. Consequently, because class II MHC molecules remain associated in class II MHC/invariant chain complexes, inhibition of cathepsin S reduces the competency of class II MHC molecules for binding antigenic peptides and reduces the presentation of antigenic peptides by class II MHC molecules.
Therefore, in one aspect, the present invention provides for methods for inhibiting;g; invariant chain proteolysis from class II MHC/invariant chain complexes, reducing the competency of class II MHC molecules for binding antigenic peptides, and reducing the presentation of antigenic peptide by class II MHC molecules, by administering to a mammalian cell, in vivo or in vitro, an amount of an inhibitor of cathepsin S effective to substantially inhibit proteolysis of invariant chain by cathepsin S.
In another aspect, because presentation of antigenic peptides complexed with class II MHC molecules is essential to immune responses which are class II MHC-restricted, the present invention also provides methods for modulating class II MHC-restricted immune responses. Such immune responses are essential to autoimmune diseases, allergic reactions, and allogeneic tissue rejections. Therefore, the present invention also provides methods for suppressing class II MHC-restricted immune responses and, in particular, autoimmune, allergic, and allogeneic immune responses, by administering to a mammal (e.g., a human patient) a therapeutically effective amount of an inhibitor of cathepsin S to reduce the presentation of antigenic peptides by class II MHC molecules and, thereby, provide a degree of relief from these conditions.
The present invention is further based, in part, upon the discovery that cathepsin S normally acts on class II MHC/invariant chain complexes at one of two, nearly adjacent, preferred invariant chain cleavage sites. In particular, it is herein disclosed that human cathepsin S normally cleaves the invariant chain at the peptide bonds C-terminal to residues Arg78 and Lys80 of the invariant chain sequence (SEQ ID NO: 1).
Therefore, in another aspect, the present invention provides for a new class of peptide-based inhibitors of cathepsin S. These peptide-based cathepsin S inhibitors are based upon the amino acid residue sequences immediately surrounding the cleavage sites which are recognized, bound, and cleaved by cathepsin S (e.g., residues 68-90, or 73-85 of SEQ ID NO: 1). The peptide-based cathepsin S inhibitors may be actual peptides or, more preferably, peptide derivatives or modified peptides which retain sufficient structural similarity to the natural substrate to retain binding activity, but which may be structurally modified to render them non-competitive inhibitors or to otherwise enhance their stability.
Preferred embodiments and exemplifications of the present invention are described in detail below.
In order to more clearly and concisely describe and disclose the subject matter of the claimed invention, the following definitions are provided for specific terms used in the specification and appended claims.
As used herein, an xe2x80x9cinhibitor of cathepsin Sxe2x80x9d is any molecular species which inhibit, the transcription of a cathepsin S gene, the processing or translation of a cathepsin S mRNA, or the processing, trafficking or activity of a cathepsin S protein, when administered in vivo or in vitro to a mammalian cell which is otherwise competent to express active cathepsin S. Thus, for example, the term xe2x80x9cinhibitor of cathepsin Sxe2x80x9d embraces a repressor which inhibits induction and/or transcription of the cathepsin S gene, or an antisense sequence which selectively binds to cathepsin S DNA or mRNA sequences and which inhibits the transcription or translation (if the cathepsin S sequences. Similarly, the term xe2x80x9cinhibitor of cathepsin Sxe2x80x9d includes competitive and non-competitive inhibitors of the activity of the cathepsin S protein, such as small molecules which structurally mimic the natural substrates of cathepsin S but which are resistant to the proteolytic activity of the enzyme. Although an inhibitor of cathepsin S may have some degree of inhibitory activity for other genes or proteins which are structurally or functionally related, the term xe2x80x9cinhibitor of cathepsin Sxe2x80x9d is not intended to embrace non-selective suppressors of all gene expression or protein synthesis, or general toxins (e.g., transcription blockers such as actinomycin D, and xcex1-amanitin, protein synthesis inhibitors such as puromycin, cycloheximide, and diptheria toxin).
As used herein, a xe2x80x9ccysteine protease inhibitorxe2x80x9d is any molecular species which inhibits one or more of the mammalian enzymes known as cysteine proteases and, in particular, which inhibits cathepsin S. The cysteine proteases, which are also known as thiol or sulfhydryl proteases or proteinases, are proteolytic enzymes with active site cysteine residues which act as nucleophiles during catalysis. Cysteine proteases include papain, calpain I, calpain II, cruzain, and cathepsins S, K, L, H, O2 and B. (Note that cathepsin D is an aspartyl protease.)
As used herein, a xe2x80x9cselective inhibitor of cathepsin Sxe2x80x9d is any molecular species which, as defined above, is an inhibitor of cathepsin S but which fails to inhibit, or inhibits to a substantially lower degree, at least one of cathepsins K, L, H, O2 and B. In preferred embodiments, a selective inhibitor of cathepsin S is employed which has a second order rate constant of inactivation or inhibition for cathepsin S which is at least twice and, more preferably, five times higher than the corresponding rate constant for at least one of cathepsins K, L, H, O2 and B. Most preferably, a selective inhibitor of cathepsin S has a second order rate constant of inactivation for cathepsin S which is at least an order of magnitude or, most preferably, at least two orders of magnitude higher than its inactivation rate constant for at least one of cathepsins K, L, H, O2 and B. As used herein, the term xe2x80x9csecond order rate constant of inactivationxe2x80x9d is intended to mean that quantity as known in the art, and represented as kinact/Ki or as k2/Ki. See, e.g., Brxc3x6mme et al., Biol. Chem. 375:343-347 (1994); Palmer et al., J. Med. Chem. 38:3193-3196 (1995); Brxc3x6mme et al., Biochem. J. 315:85-89 (1996).
As used herein, a xe2x80x9cspecific inhibitor of cathepsin Sxe2x80x9d is any molecular species which, as defined above is an inhibitor of cathepsin S but which fails to inhibit, or inhibits to a substantially lower degree each of cathepsins K, L, H, O2 and B. In preferred embodiments, a specific inhibitor of cathepsin S is employed which has a second order rate constant of inactivation for cathepsin S which is at least twice and, more preferably, five times higher than the corresponding rate constants for each of cathepsins K, L, H, O2 and B. Most preferably, a specific inhibitor of cathepsin S has a second order rate constant of inactivation for cathepsin S which is at least an order of magnitude or, most preferably, at least two orders of magnitude higher than its second order rate constants for each of cathepsins K, L, H, O2 and B.
As used herein with respect to class II MHC-restricted immune responses, xe2x80x9csuppressingxe2x80x9d means reducing in degree or severity, or extent or duration, the overt manifestations of the immune response including, for example, reduced binding and presentation of antigenic peptides by class II MHC molecules, reduced activation of T-cells and B-cells, reduced humoral and cell-mediated responses and, as appropriate to the particular immune response, reduced inflammation, congestion, pain, or necrosis. xe2x80x9cSuppressionxe2x80x9d of an immune response does not require complete negation or prevention of any of these manifestations of an immune response, but merely a reduction in degree or severity, or extent or duration, which is of clinical or other practical significance.
As used herein with respect to inhibitors of cathepsin S, the terms xe2x80x9cpeptide-basedxe2x80x9d and xe2x80x9cnon-peptide-basedxe2x80x9d do not mean that an inhibitor does, or does not, comprise a peptide or polypeptide, but that the structure of the inhibitor is based upon, or is not based upon, the structure of a polypeptide sequence which binds as a substrate in the active site of cathepsin S.
As noted above, and as evidenced in the examples below, cathepsin S is believed to be important to normal proteolytic processing of the invariant chain. Therefore, in one aspect, the present invention provides methods for inhibiting invariant chain proteolysis from class II MHC/invariant chain complexes in mammalian cells, in vivo or in vitro, by administering an of cathepsin S to the cells. As a result of the inhibition of cathepsin S, proteolysis of the invariant chain from class II MHC/invariant chain complexes within the cells is also inhibited.
Furthermore, under normal physiological conditions, inhibition of the proteolysis of the invariant chain inhibits formation of class II MHC/CLIP (also referred to as xcex1xcex2-CLIP) complexes. Therefore, as class II MHC/CLIP complexes are more readily loaded with antigenic peptides than class II MHC/invariant chain complexes, the inhibition of cathepsin S and consequent inhibition of MHC/CLIP complex formation reduces the competency of class II MHC molecules for binding antigenic peptides. Therefore, in one aspect, the present invention provides methods for reducing the competency of class II MHC molecules for binding antigenic peptides in mammalian cells, in vivo or in vitro, by administering an inhibitor of cathepsin S to the cells. As a result of inhibition of cathepsin S, proteolysis of the invariant chain is inhibited, formation of class II MHC/CLIP complexes is inhibited, and the competency of the MHC molecules to bind antigenic peptides is reduced.
Similarly, under normal physiological conditions, inhibition of the proteolysis of the invariant chain inhibits the loading and presentation of class II MHC molecules with antigenic peptides. Thus, as class II MHC/CLIP complexes are more readily loaded with antigenic peptides than class II MHC/invariant chain complexes, the inhibition of cathepsin S and consequent inhibition of MHC/CLIP complex formation reduces the loading and presentation of antigenic peptide by class II MHC molecules. Therefore, in one aspect, the present invention provides methods for reducing the presentation of antigenic peptides by class II MHC molecules in mammalian cells, in vivo or in vitro, by administering an inhibitor of cathepsin S to the cells. As a result of inhibition of cathepsin S, proteolysis of the invariant chain is inhibited, formation of class II MHC/CLIP complexes is inhibited, and the loading and presentation of antigenic peptides by class II MHC molecules is reduced.
When employed with mammalian cells in vitro, such methods have utility in preventing the loading of MHC molecules with antigenic peptides and in the production of empty MHC molecules. Empty MHC molecules have utility for subsequent loading and use as analytical, diagnostic and therapeutic agents. Alternatively, by exposing such cells in culture to high concentrations of desired antigenic peptides, or precursors of such peptides, a large proportion of MHC molecules loaded with the desired peptides may be produced. Class II MHC molecules selectively loaded with particular peptides also have utilities in analytical, diagnostic and therapeutic applications.
In these methods, an inhibitor of cathepsin S is administered to, provided to, or contacted with the cells in any manner which allows the inhibitor to enter the cells and inhibit cathepsin S. When employed in vitro, the inhibitors are typically added to the cell culture medium, although microinjection may be employed if desired. When employed in vivo, the inhibitors may be administered as described below in relation to the therapeutic methods.
As noted above, and as evidenced in the examples below, cathepsin S is believed to be important to important to normal class II MHC-restricted immune responses in mammals. Therefore, in one aspect, the present invention provides methods for suppressing class II MHC-restricted immune responses in mammals by administering an inhibitor of cathepsin S to the mammal. As a result of cathepsin S inhibition, the proteolysis of invariant chains, formation of class II MHC/CLIP complexes, and loading and presentation of antigenic peptides are inhibited and, therefore, class II MHC-restricted immune response is suppressed.
In one series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, autoimmune disease. By autoimmunity is meant the phenomenon in which the host""s immune response is turned against its own constituent parts, resulting in pathology. Many human autoimmune diseases are associated with certain class II MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class II MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host""s class II MHC molecules when complexed with peptides derived from the host""s own gene products. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention. Such autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves"" disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto""s thyroiditis.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses. By xe2x80x9callergic responsexe2x80x9d is meant the phenomenon in which the host""s immune response to a particular antigen is unnecessary or disproportionate, resulting in pathology. Allergies are well known in the art, and the term xe2x80x9callergic responsexe2x80x9d is used herein in accordance with standard usage in the medical field. Examples of allergies include, but are not limited to, allergies to pollen, xe2x80x9cragweed,xe2x80x9d shellfish, domestic animals (e.g., cats and dogs), bee venom, and the like. Another particularly contemplated allergic response is that which causes asthma. Allergic responses may occur, in part, because T cells recognize particular class II MHC/antigenic peptide complexes. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention. Although it is not expected that immunosuppression by the methods of the present invention will be a routine prophylactic or therapeutic treatment for common allergies, severe or life-threatening allergic responses, as may arise during asthmatic attacks or anaphylactic shock, may be treated according to these methods.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class II MHC genotypes (HLA types) of the donor and recipient, there may be a severe xe2x80x9callogeneicxe2x80x9d immune response against the donor tissues which results from the presence of non-self or allogeneic class II MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class II MHC/antigenic peptide complexes, inhibition of cathepsin S may suppress this response and mitigate the tissue rejection. An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective.
In order to minimize the potential for undesired side effects, it is preferred in each of the above-described embodiments that an inhibitor of cathepsin S is chosen which is a selective inhibitor of cathepsin S, a specific inhibitors of cathepsin S, or a highly specific inhibitor of cathepsin S. Thus, for example, it may not be desirable to inhibit, even briefly, all proteases or all cysteine proteases in a cell or an organism because normal protein processing and turnover will be disrupted. To the extent that such incidental inhibition is deleterious or undesired, the use of increasingly more selective cathepsin S inhibitors may be preferred. The use of more selective cathepsin S inhibitors may, for example, allow for the use of higher dosages or more extended treatment periods.
Administration of the inhibitor can be accomplished by any method which allows the inhibitor to reach the target cells, e.g., class II MHC antigen presenting cells. These methods include, e.g., injection, infusion, deposition, implantation, anal or vaginal supposition, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells by the inhibitor is obtained. Injections can be, e.g., intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal. For example, the inhibitor can be injected intravenously or intramuscularly for treatment of multiple sclerosis, or can be injected directly into the joints for treatment of arthritic disease, or can be injected directly into the lesions for treatment of pemphigus vulgaris. In certain embodiments, the injections can be given at multiple locations. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused or partially fused pellets. Inhalation includes administering the inhibitor with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the inhibitor is encapsulated in liposomes. Topical administration can be accomplished with, e.g., ointments, creams or lotions, which are applied topically to the affected area of the skin. In such compositions, the inhibitor can, e.g., be dissolved in a solvent, and then mixed with, e.g., an emulsion or a gelling agent, as are well known to persons ordinarily skilled in the art.
In certain embodiments of the invention, the administration can be designed so as to result in sequential exposures to the inhibitor over some time period, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the inhibitor by one of the methods described above, or alternatively, by a controlled release delivery system in which the inhibitor is delivered to the mammal over a prolonged period without repeated administrations. By a controlled release delivery system is meant that total release of the inhibitor does not occur immediately upon administration, but rather is delayed for some time period. Release can occur in bursts or it can occur gradually and continuously. Administration of such a system can be, e.g., by long acting oral dosage forms, bolus injections, transdermal patches and subcutaneous implants.
Examples of systems in which release occurs in bursts include, e.g., systems in which the inhibitor is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to specific stimuli, e.g., temperature, pH, light or a degrading enzyme, and systems in which the inhibitor is encapsulated by an ionically-coated microcapsule with a microcapsule core degrading enzyme. Examples of systems in which release of the inhibitor is gradual and continuous include, e.g., erosional systems in which the inhibitor is contained in a form within a matrix, and diffusional systems in which the inhibitor permeates at a controlled rate, e.g., through a polymer. Such sustained release systems can be, e.g., in the form of pellets or capsules.
The inhibitor can be suspended in a liquid, e.g., in dissolved form or colloidal form. The liquid can be a solvent, partial solvent or nonsolvent. In many cases water or an organic liquid can be used.
The inhibitor is administered to the mammal in a therapeutically effective amount. By therapeutically effective amount is meant that amount which is capable of at least partially preventing, reversing, reducing, decreasing, ameliorating or otherwise suppressing the particular immune response being treated. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of mammal; the mammal""s age, sex, size, and health; the inhibitor used; the type of delivery system used; the time of administration relative to the severity of the disease; and whether a single, multiple, or controlled release dose regimen is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
Preferably, the concentration of the inhibitor if administered systematically is at a dose of about 1.0 mg to about 2000 mg for an adult of 70 kg body weight, per day. More preferably, the dose is about 10 mg to about 1000 mg/70 kg/day. Most preferably, the dose is about 100 mg to about 500 mg/70 kg/day. Preferably, the concentration of the inhibitor if applied topically is about 0.1 mg to 500 mg/gm of ointment, more preferably is about 1.0 mg to about 100 mg/gm ointment, and most preferably is about 30 mg to about 70 mg/gm ointment. The specific concentration partially depends upon the particular inhibitor used, as some are more effective than others. The dosage concentration of the inhibitor that is actually administered is dependent at least in part upon the particular immune response being treated, the final concentration of inhibitor that is desired at the site of action, the method of administration, the efficacy of the particular inhibitor, the longevity of the particular inhibitor, and the timing of administration relative to the severity of the disease. Preferably, the dosage form is such that it does not substantially deleteriously affect the mammal. The dosage can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
The present invention employs inhibitors of cathepsin S in a variety of methods, as described above, and also provides for a new class of novel peptide-based cathepsin S inhibitors. Therefore, the present invention provides for the use of an inhibitor of cathepsin S in a medicament, or in a pharmaceutical or therapeutic preparation, for inhibiting invariant chain proteolysis from class II MHC/invariant chain complexes, for reducing the competency of class II MHC molecules for binding antigenic peptides, for reducing presentation of antigenic peptide by class II MHC molecules, for suppressing a class II MHC-restricted immune response, or for treating an autoimmune disease, allergic response, or allogeneic immune response.
The inhibitors of cathepsin S, as used in the methods of the present invention, may be regarded as being prior art inhibitors of cathepsin S, the novel peptide-based inhibitors of cathepsin S disclosed herein, or currently unknown or undisclosed inhibitors of cathepsin S which, for purposes of the present invention, are equivalents to the prior art and presently disclosed inhibitors. Alternatively, and as discussed below, inhibitors of cathepsin S may be regarded broadly as being non-peptide-based inhibitors or peptide-based inhibitors, as defined above.
A Non-Peptide-Based Inhibitors
Non-peptide-based inhibitors of cathepsin S include repressors, antisense sequences, and non-peptide-based competitive and non-competitive inhibitors.
At present, there are no known repressors of cathepsin S induction or transcription which satisfy the definition of an inhibitor of cathepsin S as used herein. Nonetheless, upon the discovery of such repressors, their use in the methods and products of the present invention may be highly preferred over currently known inhibitors, and would be regarded as an equivalent embodiment of the disclosed methods and products.
Antisense sequences to cathepsin S may readily be chosen and produced by one of ordinary skill in the art on the basis of the known nucleic acid sequence of the cathepsin S gene (see, e.g., GenBank Accession Nos. M86553, M90696, S39127; and Wiedersranders et al., J Biol. Chem. 267: 13708-13713 (1992)) and the developing field of antisense technology In order to be sufficiently selective and potent for cathepsin S inhibition, such cathepsin S-antisense oligonucleotides should comprise at least 10 bases and, more preferably, at least 15 bases. Most preferably, the antisense oligonucleotides comprise 18-20 bases. Although oligonucleotides may be chosen which are antisense to any region of the cathepsin S gene or mRNA transcript, in preferred embodiments the antisense oligonucleotides correspond to the N-terminal or translation initiation region of the cathepsin S mRNA, or to mRNA splicing sites. In addition, cathepsin S antisense may, preferably, be targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. (1994) Cell. Mol. Neurobiol. 14(5):439-457) and at which proteins are not expected to bind.
As will be obvious to one of ordinary skill in the art, the cathepsin S-inhibiting antisense oligonucleotides of the present invention need not be perfectly complementary to the cathepsin S gene or mRNA transcript in order to be effective. Rather, some degree of mismatches will be acceptable if the antisense oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides should have sufficient length and complementarity so as to selectively hybridize to a cathepsin S transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal. In addition, although it is not recommended, the cathepsin S-antisense oligonucleotides may have one or more non-complementary sequences of bases inserted into an otherwise complementary cathepsin S-antisense oligonucleotide sequence. Such non-complementary sequences may loop out of a duplex formed by a cathepsin S transcript and the bases flanking the non-complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an apparently low percentage of complementarity.
The cathepsin S-antisense oligonucleotides of the invention may be composed of deoxyribonucleotides, ribonucleotides, or any combination thereof. The 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleotide linkage. These oligonucleotides may be prepared by art recognized methods such as phosphoramidate, H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal (ed.) Meth. Mol. Biol., Humana Press, Totowa, N.J. (1993) Vol. 20; and U.S. Pat. No. 5,149,798) which may be carried out manually or by an automated synthesizer (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158).
The cathepsin S-antisense oligonucleotides of the invention also may include modified oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not compromise their ability to hybridize to nucleotide sequences contained within the transcription initiation region or coding region of the cathepsin S gene. The term modified oligonucleotide as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide. The most preferred synthetic linkages are phosphorothioate linkages. Additional preferred synthetic linkages include alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Oligonucleotides with these linkages or other modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Uhlmann et al. (1990) Chem. Rev. 90:534-583; Agrawal et al. (1992) Trends Biotechnol. 10:152-158; Agrawal.(ed.) Meth. Mol. Biol., Humana Press, Totowa, N.J. (1993) Vol. 20).
The term modified oligonucleotide also encompasses oligonucleotides with a modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having the sugars at the most 3xe2x80x2 and/or most 5xe2x80x2 positions attached to chemical groups other than a hydroxyl group at the 3xe2x80x2 position and other than a phosphate group at the 5xe2x80x2 position. Other modified ribonucleotide-containing oligonucleotides may include a 2xe2x80x2-O-alkylated ribose group such as a 2xe2x80x2-O-methylated ribose, or oligonucleotides with arabinose instead of ribose. In addition, unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
Such modifications may be at some or all of the internucleoside linkages, at either or both ends of the oligonucleotide, and/or in the interior of the molecule (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158 and Agrawal (ed.) Meth. Mol. Biol., Humana Press, Totowa, N.J. (1993) Vol. 20). Also considered as modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3xe2x80x2 and/or 5xe2x80x2 end(s) and/or various other structural modifications not found in vivo without human intervention. Other modifications include additions to the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose.
Other non-peptide-based inhibitors of cathepsin S include antibodies, including fragments of antibodies such as Fc, which selectively bind to and inhibit the activity of cathepsin S; and ribozymes which interfere with the transcription, processing or translation of cathepsin S mRNA.
B. Peptide-Based Inhibitors: Generally
Peptide-based inhibitors of cathepsin S are, at the molecular level, mimics or analogs of at least a portion of a natural polypeptide sequence which binds to the active site of cathepsin S as a substrate. In their simplest form, peptide-based inhibitors of cathepsin S are simply peptides which are based upon the sequences adjacent to known cathepsin S cleavage sites. Such peptide-based inhibitors are competitive inhibitors. Preferably, however, peptide-based inhibitors have modified polypeptide structures (whether synthesized from peptides or not) which alter their activity, stability, and/or specificity. The art of combinatorial chemistry has progressed significantly in the design of peptide-based inhibitors such that it is now routine to produce large numbers of inhibitors based on one or a few peptide sequences or sequence motifs (see, e.g., Brxc3x6mme et al., Biochem. J. 315:85-89 (1996), Palmer et al., J. Med. Chem. 38:3193-3196. (1995)). Thus, for example, if cathepsin S is known to cleave protein X at position Y, a peptide-based inhibitor of cathepsin S may be chosen or designed as a polypeptide or modified polypeptide having the same sequence as protein X, or structural similarity to the sequence of protein X, in the region adjacent to position Y. In fact, the region adjacent to the cleavage site Y, spanning residues removed by 10 residues or, more preferably, five residues N-terminal and C-terminal of position Y, may be defined as a xe2x80x9cpreferred protein X cleavage sitexe2x80x9d for the choice or design of peptide-based inhibitors. Thus, a plurality of peptide-based inhibitors, chosen or designed to span the preferred protein X cleavage site around position Y, may be produced, tested for inhibitory activity, and sequentially modified to optimize or alter activity, stability, and/or specificity.
Preferably, the peptide portion of the peptide-based inhibitors of the invention can be any length, as long the compound can inhibit proteolysis by cathepsin S. Preferably, the peptide portion is about 2 to about 20 amino acids or amino acid equivalents long, more preferably it is about 2 to about 10 amino acids or amino acid equivalents long, and most preferably it is about 2 to about 3 amino acids or amino acid equivalents long.
Modified peptide-based inhibitors of cysteine proteases, as well as other enzymes, are well known in the art. Thus, for example, peptide-based inhibitors of cysteine proteases include peptidyl aldehydes, nitrites, xcex1-ketocarbonyls, halomethyl ketones, diazomethyl ketones, (acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts, epoxides, and N-peptidyl-O-acylhydroxylamines (see, e.g., Brxc3x6mme et al., Biochem. J. 315:85-89 (1996); Palmer et al., J. Med. Chem. 38(17):3193-3196 (1995); Brxc3x6mme et al., Biol. Chem. 375:343-347 (1994); and references cited therein.
Currently preferred peptide-based inhibitors of cathepsin S include those based upon the sequences Leu-Leu-Leu, and Leu-Hph (where Hph indicates homophenylalanine), as well as the preferred invariant chain cleavage site sequences described below. Thus, for example, morpholinurea-Leu-Hph-vinylsulfone phenyl (LHVS) is one preferred cathepsin S inhibitor. Also preferred are other peptidyl vinyl sulfones, with or without the addition of an N-terminal groups such as carboxybenzyl or nitrophenylacetyl groups, such as Leu-Leu-Leu-vinyl sulfone, N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone, and N-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone. Most preferably, the peptidyl moiety corresponds to 2-3 residues chosen from the cathepsin S preferred invariant chain cleavage site, as described below.
C. Peptide-Based Inhibitors: Novel Cathepsin S Inhibitors
As noted above, and evidenced in the Examples below, cathepsin S cleaves the invariant chain, while associated in a class II MHC/invariant chain complex, at two major, nearly adjacent, locations. These major cleavages occur C-terminal of the Arg residue at position 78 or the Lys residue at position 80 of the human invariant chain sequence (SEQ ID NO: 1). See Example 8. Therefore, a preferred invariant chain cleavage site extends around Arg78 from N-terminally about positions 68-73 to C-terminally from about positions 83-88. Similarly, a preferred invariant chain cleavage site extends around Lys80 from N-terminally about positions 70-75 to C-terminally from about positions 85-90. Because of the overlap of these regions, cathepsin S has a preferred invariant chain cleavage site spanning, approximately, from N-terminally about positions 68-75 to C-terminally about positions 83-90. Thus, peptide-based inhibitors of cathepsin S based upon a sequence of 2-20, more preferably 2-10, and most preferably 2-3 consecutive residues from within this site are provided.
Because of the postulated nature of the cathepsin S active site (see, e.g., Brxc3x6mme et al., Biochem. J. 315:85-89 (1996)), it is particularly preferred that the residues one-, two- and, optionally, three-positions N-terminal to the cleavage sites be included in a peptide based inhibitor. Thus, for example, the residues Asn-Leu (positions 76 and 77; or xe2x88x922 and xe2x88x921 relative to the Arg78 cleavage point) or the residues Glu-Asn-Leu (positions 75, 76, and 78; or xe2x88x923, xe2x88x922 and xe2x88x921 relative to the Arg78 cleavage point) are preferably used as a basis for choosing or designing a peptide-based inhibitor. Similarly, the residues Arg-Met (positions 78 and 79; or xe2x88x922 and xe2x88x921 relative to the Lys80 cleavage point) or the residues Leu-Arg-Met (positions 77, 78, and 79; or xe2x88x923, xe2x88x922 and xe2x88x921 relative to the Lys80 cleavage point) are preferably used as a basis for choosing or designing a peptide-based inhibitor.
Thus, for example, the invention provides novel peptide-based inhibitors such as vinylsulfones compounds including Asn-Leu-vinylsulfone, Arg-Met-vinylsulfone, Leu-Arg-Met-vinylsulfone, Glu-Asn-Leu-vinylsulfone, and Leu-Leu-Leu-vinylsulfone. Modifications of these peptide vinylsulfones are also included in the invention. For example, carboxybenzyl can be present at the N-terminal end to give the following compounds: N-(carboxybenzyl)-Asn-Leu-vinylsulfone, N-(carboxybenzyl)-Arg-Met-vinylsulfone, N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone, N-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone, and N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone. In an alternative, nitrophenylacetyl is present at the N-terminal end to give the following compounds: N-(nitrophenylacetyl)-Asn-Leu-vinylsulfone, N-(nitrophenylacetyl)-Arg-Met-vinylsulfone, N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone, N-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone, and N-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone. A peptide-based vinylsulfone is meant to include, e.g., a peptide vinylsulfone or a modified peptide vinylsulfone. The invention is also meant to include other modifications of the peptide-based vinylsulfones, e.g., substitutions can be added at the amino terminus of the peptide-based vinylsulfones by, e.g., N-methyl substituents or any other alkyl or substituted alkyl chain, or by substitution with, e.g., phenyl, benzyl, aryl, or modified aryl substituents, as would be known to those skilled in the art.
These examples are merely illustrative and not exhaustive. For example, the peptide-based inhibitors of cathepsin S can be based upon other peptide sequences which span a portion of the preferred invariant chain cleavage site, for example, any 2-3, 3-5, 5-7, or more consecutive residues within the preferred invariant chain cleavage site. Similarly, the peptide-based inhibitors may be peptidyl aldehydes, nitriles, xcex1-ketocarbonyls, halomethyl ketones, diazomethyl ketones, (acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts, epoxides, N-peptidyl-O-acylhydroxylamines, or other such compounds known to those of skill in the art. Furthermore, the N-termini of these peptide-based inhibitors of cathepsin S may be blocked with a variety of substituent groups, including N-methyl substituents or other alkyl or substituted alkyl chains; phenyl, benzyl, aryl, or modified aryl substituents; or other such substituents known to those of skill in the art.